Method of carrying out preparative thin-layer chromatography and apparatus for use in the method

ABSTRACT

A method of carrying out preparative thin-layer chromatography comprising application of a layer of adsorbent material to a base plate, application of a sample in the form of a narrow strip in the adsorbent layer and immersion of the base plate in a solvent for the development of the chromatogram is disclosed wherein the sample is applied in solid form, the sample being dispensed in a groove in the adsorbent layer to form a strip which is then compressed or compacted. The invention also comprises apparatus for carrying out the method according to the invention which apparatus includes (a) a bridge member, (b) a holder for cutting blades, (c) an applicator and (d) a press of T-shaped crosssection.

United States Patent [191 Meyer METHOD OF CARRYING OUT PREPARATIVETHIN-LAYER CHROMATOGRAPHY AND APPARATUS FOR USE IN THE METHOD [76]Inventor: Holger Meyer, Veerstucken 5j,

D-2000 Hamburg 60, Germany [22] Filed: Dec. 20, 1974 [21] Appl. No.:534,916

OTHER PUBLICATIONS Thin-Layer Chromatography An Annotated Bibliography:1964-1968, by Haywood, B. J., Ann Arbor Science Publishers, Inc., AnnArbor, Mich., 1968, pp.

6-34 relied on.

[451 Oct. 28, 1975 Primary ExaminerJohn Adee Attorney, Agent, orFirmFred Philpitt [57] ABSTRACT A method of carrying out preparativethin-layer chromatography comprising application of a layer of adsorbentmaterial to a base plate, application of a sample in the form of anarrow strip in the adsorbent layer and immersion of the base plate in asolvent for the development of the chromatogram is disclosed wherein thesample is applied in solid form, the sample being dispensed in a groovein the adsorbent layer to form a strip which is then compressed orcompacted. The invention also comprises apparatus for carrying out themethod according to the invention which apparatus includes (a) a bridgemember, (b) a holder for cutting blades, (c) an applicator and (d) apress of T-shaped cross-section.

12 Claims, 6 Drawing Figures Sheet 1 of 3 3,915,856

US. Patent Oct. 28, 1975 Sheet 2 of 3 3,915,856

US. Patent Oct. 28, 1975 US. Patent 'Oct. 28, 1975 Sheet 3 of3 3,915,856

METHOD OF CARRYING OUT PREPARATIVE THIN-LAYER CHROMATOGRAPHY ANDAPPARATUS FOR USE IN THE METHOD The present invention relates to amethod of carrying out preparative thin-layer chromatography comprisingthe application of a layer of adsorbent material to a base plate,application of a sample in a narrow strip in the adsorbent layer andimmersion of the base plate in a solvent for the development of thechromatogram.

Hitherto, in preparative chromatography, the sample has been applied tothe adsorbent layer in the form of a solution. When doing this, thesample has generally been applied in the form of a solution in water orin an organic solvent in a narrow strip on the adsorbent layer by meansof an injection-syringe provided with a capillary. Among thedisadvantages of this way of application the following may be mentioned:

l. The dissolved state of the sample renders it difficult to apply adefined dosage.

2. In view of the facts that the initial strip must be at most 2 to 3 mmin width and that the sample should be evenly distributed along thestrip the application requires a comparatively long time, typically atleast 20 minutes. Moreover, the solvent has to evaporate before asubsequent strip can be applied.

3. When applying a solution most of the sample is sucked up into thesurface layer of the adsorbent and the concentration distribution of theinitial strip is liable to become non-uniform in length, width anddepth.

It has now been found possible to avoid or alleviate these disadvantagesby means of a method which is characterized in that the sample isapplied in solid form, the sample being dispensed in a groove in theadsorbent layer to form a strip which is then compressed. The sample isapplied either in pure form or in homogeneous mixture with adsorbentmaterial in various weight ratios. The advantages gained by using thismethod are primarily that a carefully weighed amount of sample may bechromatographed and that the sample strip in the adsorbent layer may bemade thoroughly homogeneous.

According to a preferred form of the method according to the invention,the sample strip and those parts of the adsorbent layer which, onimmersion of the base plate in the solvent, are adjacent to or below thesample strip, are held against the base plate during the development ofthe chromatogram.

The present invention also provides apparatus for carrying out themethod acoording to the invention, which apparatus includes a. a bridgehaving longitudinal, high sides and a bottom which is provided with anarrow longitudinal slot,

b. a holder for cutting blades, which holder is designed for cutting outa groove in the adsorbent layer, following the slot of the bridge,

c. an applicator designed for dispensing the sample into the groove cutin the adsorbent layer and d. a press of T-shaped cross-section, thestem of which fits into the slot of the bridge for compressing thesample in the groove.

Preferably, the cutting blade holder is provided with means for scrapingthe groove in the adsorbent layer to remove adsorbent material when saidholder is moved to and fro in the bridge.

Preferably, the interior of the applicator has the shape of a funnelhaving an outlet at the base thereof,

said outlet being arranged to register with the slot of the bridge whenthe applicator is placed between the longitudinal high sides of thebridge. It is also of advantage to provide the applicator with twowedge-shaped levelling means on opposite sides of the outlet forlevelling the sample in the groove.

The T-stem of the T-shaped press is preferably somewhat longer than thethickness of the bottom of the bridge. The press is also advantageouslyprovided with finger grips.

Apparatus according to the present invention may also include:

e. a trough having vertical side and end walls and a bottom, at leastone of the side walls being perforated near said bottom,

f. an insert member, which rests on the bottom of the trough and isshaped to provide a plurality of equally high elevations and g. meansfor securing the base plate with the adsorbent layer applied thereon inclose contact with one of the side walls of the trough.

The invention will now be illustrated, by way of example, with referenceto the accompanying drawings, wherein FIGS. 1 3 illustrate differentsteps of the method according to the invention,

FIG. 4 is a view in perspective of one form of applicator for apparatusaccording to the invention,

FIG. 5 is a view in perspective of one form of blade holder forapparatus according to the invention and FIG. 6 illustrates analternative to the step of FIG. 3.

In FIG. 1 there is shown a frame plate 1 comprising aluminium, stainlesssteel or any other material which will allow heating of a glass platewith adsorbent thereon lying on said frame plate to a suitabletemperature, usually C, for a sufficiently long time to activate theadsorbent, i.e., to remove completely water and the liquid in which theadsorbent was suspended when the thin layer plate was prepared. Theframe plate 1 is provided with border members 2 along two parallel edgesand a third somewhat higher and removable border member 3 at rightangles to the border members 2. A base plate 4 is located within theborders, and may comprise glass or any other material which is resistantto prolonged heating to about 120C and to the solvents in which theplate is immersed for the development of the chromatogram. The baseplate 4 is placed on flat rods 5 of aluminium, stainless steel or asimilar material laid out along the borders 2 in such a number that theupper surface of the plate 4 will be at such a vertical distance fromthe upper surface of the borders 2 as is required to accommodate thethickness of the adsorbent layer. An adsorbent layer 6 is applied to thebase plate 4. This layer may, for instance, be produced by pouring ontothe plate a suspension of silica gel or another adsorption agent inwater or other suitable liquid, and levelling the suspension to form auniformly thick layer by means of a rod 7 laid on the borders 2 at rightangles thereto and drawn along said borders 2 to distribute thesuspension into a layer of a welldefined uniform thickness on the plate4. The layer is then allowed to dry, after which it is activated atabout 120C for 1 2 hours to remove the suspending medium. This alsoproduces a small decrease in the thickness of the layer. Prepared plateswith an adsorbent layer already applied thereon are commerciallyavailable and may also be used for the method according to theinvention.

After the adsorbent layer 6 has been prepared a bridge 8 having two highlongitudinal sides 9 and a bottom 10 with a longitudinal slot 1 l isplaced on the borders 2. The bridge is suitably provided with downwardlyprojecting pegs (not shown) which should engage into notches 12 in theborders 2. The bridge is suitably also chamfered at the ends thereof sothat the unchamfered middle part of the bottom side is given the samelength as the inside distance between the two borders 2 of the frameplate 1. In this way, the bottom side of the bridge 8 can lie close tothe adsorbent layer. The slot 11 is situated about 10 mm from the edgeof the layer.

From FIG. 2 it can be seen that the bridge 8 is secured to the frameplate 1 by fastening means comprising screw clamps 13. In this positiona groove is cut in the adsorbent layer 6 by means of cutting bladesmounted in a holder, for instance a holder of the type shown in FIG. 5.

The holder 14 shown in FIG. 5 comprises a central portion 15 and twoside portions 16 held together with screws 17. Two cutting blades 18 areheld by means of the screws between the central portion 15 and the sideportions 16, one blade between each side portion 16 and the centralportion 15, in such a position that the edges thereof will be at adistance from the bottom side of the central and side portionscorresponding to the desired depth of the groove in the adsorbent layer.The distance between the outside surfaces of the cutting blades 18corresponds to the width of the slot 11 of the bridge 8 and the overallwidth of the holder is equal to the internal distance between thelongitudinal vertical sides 9 of the bridge. Tongues 19 are arrangedbetween the cutting blades 18.

When cutting out a groove in the adsorbent layer the blade holder 14 isplaced between the sides 9 of the bridge in such a way that the cuttingblades 18 are inserted in the slot 11. Then the holder 14 is moved toand fro in the slot along the longitudinal axis of the bridge, and theblades 18 cut the outer edges of a groove in the adsorbent layer 6,which groove is scraped clean by the tongues 19.

Then the blade holder, and loose adsorbent material possibly remainingin the groove, are removed and the sample is introduced into the grooveby means of an applicator which may, for example, be as shown in FIG. 4.

The interior of the applicator 20 shown in FIG. 4 forms a funnel 21ending in a rectangular slot-shaped outlet 22 in the base of theapplicator. Two wedges 23 of the same breadth as the slot 11 of thebridge 8 are arranged at the base of the applicator at the ends of theoutlet 22. In operation, the applicator is placed between the sides 9 ofthe bridge 8 in such a way that the wedges 23 engage in the slot 11. Asolid sample (which may be pure or mixed with adsorbent) is charged intothe funnel 21 and the applicator is moved to and fro along the slot 11.During this operation the sample flows out through the outlet 22 and isdistributed in the groove in the adsorbent layer where it is levelled bymeans of the wedges 23. The maximum thickness of the wedges 23 isadapted to the surface of the adsorbent layer in such a way that thesurface of the levelled sample in the groove will be of about the samelevel as the surface of the adsorbent layer.

After the sample has been distributed into and levelled in the groovethe applicator is removed and the sample is compressed by using aT-shaped press 24 (FIG. 2), the T-stem 25 of which fits into the slot 11of the bridge. The upper part 26 of the press is of the same width asthe internal distance between the longitudinal sides 9 of the bridge andis provided with two finger grips 27 for facilitating its insertion intothe bridge and its removal therefrom. The stem 25 is somewhat longerthan the thickness of the bottom 10 of the bridge.

After compression of the sample, the press 24 is removed and the bridge8 and the plates 1 and 4 may if desired be immersed in a solvent for thedevelopment of the chromatogram. In order, however, to avoid flaking ofthat part of the adsorbent layer which on immersion of the plates intothe solvent is situated beneath the strip of sample, one or other of theexpedients illustrated in FIGS. 3 and 6, respectively, may suitably beadopted.

In FIG. 3 the removable border 3 (vide FIGS. 1 and 2) of the frame plate1 has been removed and a sheet 28 of a porous material which does notcontaminate the sample, the adsorbent layer or the solvent, for instancea filter paper, has been folded round the adsorbent layer 6 and the baseplate 4 at the edge thereof which is nearest to the sample strip 29. Thesheet 28 extends sufficiently far up the adsorbent layer that it coversthe strip 29 of sample. A plate 30, made of glass or of any othermaterial that is resistant to the solvent, is placed outside the sheet28 between the borders 2, and is secured by fastening means, in thiscase the same screw clamps 13 as were used earlier for securing thebridge 8 to the frame plate 1 (FIG. 2).

Instead, as shown in FIG. 6, the base plate 4 may be completely removedfrom the frame plate and placed in a trough 31 having vertical side andend walls 32 and 33, respectively, and a bottom 34. The side walls 32are perforated with apertures 35 situated near the bottom 34. The bottom34 has an insert in the form of a slightly corrugated metal sheet 36resting thereon, said sheet presenting a large number of points ofsupport for the base plate 4 and the adsorbent layer 6 applied thereon.The base plate is placed in such a way that the adsorbent layer 6 is inclose contact with one of the side walls 32 of the trough. The plate issecured in this position by fastening means comprising screws 37 passingthrough the opposite side wall. The height of the trough is such thatthe strip 29 of sample will lie below the top edge of the trough whenthe base plate 4 is resting on the sheet 36. The distance between thestrip 29 of sample and the top edge of the trough suitably amounts toabout 5 mm or possibly somewhat more. The apertures 35 may, forinstance, have a diameter of 1 mm and be situated about 5 mm above thebottom 34 and about 1 cm apart. The trough is suitably made a millimeteror so longer than the width of the plate.

For the development of the chromatrogram, the base plate with the troughattached thereto is immersed in a solvent and the development may thenbe carried out in conventional manner.

It is easily recognized that the embodiment in detail of the componentsforming parts of the invention may be performed in many ways withoutdeparting from the idea of the invention. For that reason the inventionis not limited to the embodiments of said components shown in thefigures of the accompanying drawings.

What is claimed is:

l. A method of carrying out preparative thin-layer chromatographycomprising application of an adsorbent layer to a base plate,application of a sample in the form of a narrow strip in the adsorbentlayer and immersion of the base plate in a solvent for the developmentof the chromatogram, characterized in that the sample is applied insolid form, the sample being dispensed in a groove in the adsorbentlayer to form a strip which is then compressed or compacted.

2. A method according to claim 1, wherein the sample is applied alone orin homogeneous admixture with adsorbent material.

3. A method according to claim 1, wherein the strip of sample and thoseparts of the adsorbent layer which, on immersion of the ground plate inthe solvent, are adjacent to or below the sample strip are held againstthe ground plate during the development of the chromatogram.

4. A method according to claim 3, wherein a sheet of a porous materialwhich does not contaminate the sample, the adsorbent layer or thesolvent is folded round the adsorbent layer at the edge thereof that isnearest to the strip of sample, care being taken that the sheet coversthe strip of sample, and the sheet is secured in this position duringthe developing of the chromatogram.

5. A method according to claim 3, wherein the base plate with theadsorbent layer applied thereon is caused to rest upon a plurality ofpoints of support at a small distance from the bottom of a trough havingvertical side and end walls and is secured in close contact with one ofthe vertical side walls with the adsorbent layer facing said one sidewall, the height of said one side wall being adapted to the position ofthe strip of sample so that said strip will be situated somewhat belowthe top edge of said wall, said wall and the opposite side wall beingperforated near the bottom of the trough.

6. Apparatus for carrying out the method according to claim 1,characterized in that it includes a. a bridge having longitudinal sidesand a bottom,

said bottom being provided with a narrow longitudinal slot,

b. a holder for cutting blades, which holder is designed for cutting outa groove in the adsorbent layer by following the slot of the bridge c.an applicator designed for dispensing the sample in the groove cut inthe adsorbent layer, and

d. a press of T-shaped cross-section, the stem of which is arranged tofit into the slot of the bridge for compressing the sample in thegroove.

7. Apparatus according to claim 6, wherein the blade holder is providedwith means for scraping clean the groove in the adsorbent layer whensaid holder is moved to and fro along the slot in the bridge.

8. Apparatus according to claim 6, wherein the interior of theapplicator has the shape of a funnel and has an outlet arranged in itsbase, said outlet being arranged to register with the slot of the bridgewhen the applicator is placed between the longitudinal sides of thebridge.

9. Apparatus according to claim 8, wherein two wedge-shaped levellingmeans are arranged on opposite sides of the outlet for levelling of thesample in the groove.

10. Apparatus according to claim 6, wherein the T- shaped press isprovided with finger grips.

11. Apparatus according to claim 6, wherein the T- stem of the T-shapedpress projects somewhat further than the thickness of the bottom of thebridge.

12. Apparatus according to claim 6, characterized in that it alsoincludes e. a trough having vertical side and end walls and a bottom, atleast one of the side walls being perforated near said bottom (34),

f. an insert which rests on the bottom of the trough and is shaped toprovide a plurality of equally high elevations, and

g. means for securing a base plate with an adsorbent layer appliedthereon in close contact with one of the side walls of the trough.

1. A METHOD OF CARRYING OUT PREPARATIVE THIN-LAYER CROMATOGRAPHYCOMPRISING APPLICATION OF AN ADSORBENT LAYER TO A BASE PLATE,APPLICATION OF A SAMPLE IN THE FORM OF A NARROW STRIP IN THE ADSORBENTLAYER AND IMMERSION OF THE BASE PLATE IN A SOLVENT FOR THE DEVELOPMENTOF THE CROMATOGRAM, CHARACTERIZED IN THAT TH SAMPLE IS APPLIED IN SOLIDFORM, THE SAMPLE BEING DISPENSED IN A GROOVE IN THE ADSORBENT LAYER TOFORM A STRIP WHICH IS THEN COMPRESED OR COMPACTED.
 2. A method accordingto claim 1, wherein the sample is applied alone or in homogeneousadmixture with adsorbent material.
 3. A method according to claim 1,wherein the strip of sample and those parts of the adsorbent layerwhich, on immersion of the ground plate in the solvent, are adjacent toor below the sample strip are held against the ground plate during thedevelopment of the chromatogram.
 4. A method according to claim 3,wherein a sheet of a porous material which does not contaminate thesample, the adsorbent lAyer or the solvent is folded round the adsorbentlayer at the edge thereof that is nearest to the strip of sample, carebeing taken that the sheet covers the strip of sample, and the sheet issecured in this position during the developing of the chromatogram.
 5. Amethod according to claim 3, wherein the base plate with the adsorbentlayer applied thereon is caused to rest upon a plurality of points ofsupport at a small distance from the bottom of a trough having verticalside and end walls and is secured in close contact with one of thevertical side walls with the adsorbent layer facing said one side wall,the height of said one side wall being adapted to the position of thestrip of sample so that said strip will be situated somewhat below thetop edge of said wall, said wall and the opposite side wall beingperforated near the bottom of the trough.
 6. Apparatus for carrying outthe method according to claim 1, characterized in that it includes a. abridge having longitudinal sides and a bottom, said bottom beingprovided with a narrow longitudinal slot, b. a holder for cuttingblades, which holder is designed for cutting out a groove in theadsorbent layer by following the slot of the bridge c. an applicatordesigned for dispensing the sample in the groove cut in the adsorbentlayer, and d. a press of T-shaped cross-section, the stem of which isarranged to fit into the slot of the bridge for compressing the samplein the groove.
 7. Apparatus according to claim 6, wherein the bladeholder is provided with means for scraping clean the groove in theadsorbent layer when said holder is moved to and fro along the slot inthe bridge.
 8. Apparatus according to claim 6, wherein the interior ofthe applicator has the shape of a funnel and has an outlet arranged inits base, said outlet being arranged to register with the slot of thebridge when the applicator is placed between the longitudinal sides ofthe bridge.
 9. Apparatus according to claim 8, wherein two wedge-shapedlevelling means are arranged on opposite sides of the outlet forlevelling of the sample in the groove.
 10. Apparatus according to claim6, wherein the T-shaped press is provided with finger grips. 11.Apparatus according to claim 6, wherein the T-stem of the T-shaped pressprojects somewhat further than the thickness of the bottom of thebridge.
 12. Apparatus according to claim 6, characterized in that italso includes e. a trough having vertical side and end walls and abottom, at least one of the side walls being perforated near said bottom(34), f. an insert which rests on the bottom of the trough and is shapedto provide a plurality of equally high elevations, and g. means forsecuring a base plate with an adsorbent layer applied thereon in closecontact with one of the side walls of the trough.